Abstract A major goal of this proposal is to define the role of the airway epithelial cells in the immune response to respiratory viral infection and to extend this information to better control acute viral illness and prevent post- viral airway disease. Based on new insights into the issue of how airway epithelial cells mediate host defense against respiratory viruses, we will develop the hypothesis that the proper quality and quantity of interferon (IFN) signaling in airway epithelial cells is critical for controlling acute viral illness and post-viral airway disease. Studies of transgenic mice and transduced human cell lines further revealed that IFN signaling is limited by a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote IFN-stimulated gene (ISG) expression and on viral 3C protease to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the IFN signaling pathway and specially controls viruses like human rhinovirus (HRV) that require viral 3C protease for replication. We therefore hypothesize that airway epithelial cell PARP9-DTX3L specially regulates the severity of infection due to HRV and other picornaviruses like human enterovirus-D68 (HEV-D68) that might be more severe in patients with airway disease and/or linked to exacerbation or progression of airway disease. We also propose that the novel dual-mechanism for PARP9-DTX3L to safely harness IFN signaling reserve provides a roadmap to protect against respiratory viral infection and post-viral airway disease. Indeed, we used our findings to develop small-molecule IFN-signal enhancer (SMISE) compounds to enable the antiviral benefit of PARP9-DTX3L expression. To further develop these insights and translate them to practice, we have the following Specific Aims: 1. In human cell and molecular models, determine the role of PARP9-DTX3L in IFN signaling and control of respiratory picornaviruses in primary-culture human airway epithelial cells and recombinant human proteins. This Aim will also pursue the mechanism for PARP9-DTX3L activities and a structural biology approach to enhancing these actions. 2. In mouse cell and in vivo models, define the role of PARP9-DTX3L in IFN signaling and control of respiratory picornaviruses and post-viral airway disease using cell-specific transgenic, knock-out, and knock-in mice and airway epithelial cells from these mice. This Aim will develop new models of picornavirus infection and pursue the mechanism for PARP9-DTX3L activities in IFN signaling and SMISE effect. Together, the results will provide the first proof-of-concept for the role of PARP9-DTX3L in controlling respiratory picornavirus infection in vitro and in vivo and will advance the basis for developing an effective antiviral compound for this type of infection.